Constable, SCJ; (2015) Investigating the role of Plzf in neural progenitors. Doctoral thesis , UCL (University College London).

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Abstract

Neurogenesis must be tightly regulated both spatially and temporally to give rise to the full spectrum of cells of the nervous system. The promyelocytic leukaemia zinc finger (Plzf) transcription factor is required for maintenance of stem cells of the spermatogonial and haematopoietic systems and is widely expressed throughout the vertebrate central nervous system. I aimed to understand whether Plzf has a role in the maintenance of neural progenitors, using the zebrafish as my model organism. To gain information about the transcriptional targets directly regulated by Plzf in zebrafish, I performed chromatin immunoprecipitation on embryos expressing epitope-tagged Plzfa. This analysis failed to identify targets and subsequent troubleshooting determined that the epitope-tagged Plzfa protein wasn’t functional. Analysis of plzfa and plzfb expression during development reveals that both genes are coexpressed in progenitors in the hindbrain, leading to the hypothesis that the two proteins are functionally redundant. In support of this hypothesis, morpholino-mediated knockdown of the two proteins resulted in a defect in progenitor maintenance. To complement this work, I introduced targeted mutations in the plzfa and plzfb genes using transcription activator-like effector nuclease (TALEN) technology. In contrast to the morpholino-based results, inactivation of these genes did not result in the same defect in progenitor maintenance; leading to the conclusion that previously unknown off-target effects associated with morpholino use caused the phenotype. Finally, I developed a simple and highly efficient technique for the insertion of exogenous DNA into targeted locations into the zebrafish genome, aided by the use of TALENs. Using this technique to analyse plzfa expression complements earlier analysis suggesting that plzfa is expressed in cells actively undergoing neurogenesis. Preliminary functional analysis supports a hypothesis that Plzfa regulates multiple steps during the neurogenic cascade and is important to ensure the correct timing of proneural gene expression during the pathway to differentiation.

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